The long term goal of this project is to understand the mechanism of intracellular membrane fusion events. In the last project period, utilizing a cell-free system that reconstitutes intercisternal transport in the Golgi stack as an assay system, we discovered and purified an N- ethylmaleimide-sensitive fusion ATPase (NSF) that is required for fusion processes involving multiple compartments in yeast and animal cells. We also found and purified three NSF attachment proteins (SNAPs) that assemble with NSF and an integral membrane SNAP receptor to form a 20S particle that seems likely to be involved in fusion processes. We propose to build upon this foundation by better characterizing the structure and function of NSF, SNAPs and snap receptor, to definitively establish their roles in fusion in living cells, and to isolate additional fusion components. We will test our hypothesis that the 20S NSF-containing particle is the "core" of a general intracellular membrane fusion machinery that assembles from dispersed subunits upon demand when a fusion event is required, and disassembles in the course of catalyzing fusion.